Wakako Tamo

Interferon-γ stimulates the expression of galectin-9 in cultured human endothelial cells

Galectin‐9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin‐9 expression in endothelial cells. Galectin‐9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon‐γ (IFN‐γ). IFN‐γ also enhanced the adhesion of human eosinophilic leukemia‐1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin‐4, which induces eotaxin expression, did not affect the expression of galectin‐9. The in situ endothelium from patients with inflammatory diseases was found to express galectin‐9. IFN‐γ‐induced production of galectin‐9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Synergistic stimulation, by tumor necrosis factor-α and interferon-γ, of fractalkine expression in human astrocytes

Fractalkine is a CX3C chemonkine that appears to be a neuron-to-microglia signal molecule in the central nervous system. We studied the expression of fractalkine in normal human astrocytes in culture, by using semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that tumor-necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically enhance the expression of fractalkine. The expression of both fractalkine mRNA and protein was increased in time- and concentration-dependent manners in the cells co-stimulated with TNF-alpha and IFN-gamma. Cycloheximide, an inhibitor of protein synthesis, and dexamethasone had no effect on the synergy of the stimulation of fractalkine expression. We conclude that normal human astrocytes produce fractalkine by co-stimulation with pro-inflammatory cytokines and it may serve as a potential signal for immune and inflammatory responses in the central nervous system.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1β (IL-1β) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 ×g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1β, and this may contribute to inflammatory reactions in the CNS.

Expression of α-synuclein, the precursor of non-amyloid β component of Alzheimer's disease amyloid, in human cerebral blood vessels

The non-amyloid β component of Alzheimers disease amyloid (NAC) is detected in cerebral amyloid angiopathy; and the precursor of NAC is now known to be identical to α-synuclein (α-S), a major component of Lewy bodies in Parkinsons disease. We studied if cerebral vascular cells express α-S. Immunohistochemical studies of human cerebral tissues from control and cerebral amyloid angiopathy patients revealed the expression of α-S in vascular endothelial and smooth muscle cells. Then we studied the expression of α-S in vitro using cultures of vascular cells. Cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells were found to constitutively express α-S messenger RNA and protein. α-S is normally expressed in vascular cells and may play some physiological role in the vascular wall.

Upregulation of α-synuclein by lipopolysaccharide and interleukin-1 in human macrophages

α‐Synuclein was originally identified as the presynaptic nerve terminal protein. Recently, we reported that α‐synuclein is also expressed in cultured human astrocytes and that its levels are increased by stimulation with interleukin‐1β, suggesting that it may be involved in inflammatory processes. We therefore investigated the effect of inflammatory stimuli on α‐synuclein expression in human macrophages. α‐Synuclein mRNA and protein were detected in cultured human macrophages and levels of α‐synuclein protein were increased by stimulation with lipopolysaccharide and interleukin‐1β in a time‐ and concentration‐dependent manner. Immunofluorescent staining showed that α‐synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, α‐synuclein immunoreactivity was present in alveolar macrophages from human lung tissues. These findings suggest that the function of α‐synuclein is not exclusive to the nervous system and that α‐synuclein may play a role in inflammatory processes and immune responses.

Relationship between postoperative recurrence and expression of cyclin E, p27, and Ki-67 in non-small cell lung cancer without lymph node metastases

Abstract.Background: Cyclin E and p27 play pivotal roles in cancer development and progression. We investigated whether the prognosis in cases of non-small cell lung cancer without lymph node metastases that underwent complete resection could be associated with tissue expression of cyclin E, p27, and Ki-67. Methods: Tumors from 62 patients at least 5 years after surgery were assessed by immunohistochemistry for expression of cyclin E, p27, and Ki-67. Disease-free survival (DFS) after surgery was used to evaluate disease prognosis. We also investigated the relationship between expression of these factors and postoperative recurrence. Results: In non-small cell lung cancer, p27-negative expression and pT factor were significantly unfavorable prognostic factors in multivariate analysis. The DFS rate in cyclin E-positive expression was significantly lower than in cyclin E-negative expression. Similarly, p27-negative expression and high Ki-67 expression correlated with a shortened DFS rate. In combinations of expression of cyclin E and p27, the cyclin E-negative/p27-positive group had a significantly higher DFS rate than did the other groups. According to histological type, there were correlations between the risk of postoperative recurrence and expression of these three biological factors, especially in adenocarcinoma. Conclusion: By analyzing the expression of cyclin E, p27, and Ki-67 of tumor cells, it was possible to extract the patient group for whom closer follow-up and postoperative treatment is necessary to improve survival rate.

Proteasome inhibitor MG-132 enhances the expression of interleukin-6 in human umbilical vein endothelial cells: Involvement of MAP/ERK kinase

Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL‐6 expression by the ubiquitin‐protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG‐132, a protease inhibitor, and the levels of IL‐6 mRNA and protein were measured by reverse transcription‐PCR and ELISA. MG‐132 increased the expression of IL‐6 mRNA and protein; and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK1/2). MG‐132 treatment was also found to enhance the level of phosphorylated MEK1/2. Treatment of the cells with actinomycin D inhibited IL‐6 expression in response to MG‐132, suggesting the transcriptional upregulation of IL‐6 under proteasomal inhibition. We conclude that a protease inhibitor MG‐132 upregulates IL‐6 expression in vascular endothelial cells, at least in part, through the activation of MEK1/2.

Platelet-activating factor enhances the expression of vascular endothelial growth factor in normal human astrocytes

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. To examine whether platelet-activating factor (PAF) induces the expression of VEGF in human astrocytes, we stimulated cultured normal astrocytes with PAF and performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR for VEGF mRNA and enzyme-linked immunosorbent assay for VEGF protein. PAF increased the expression of VEGF in astrocytes in time- and dose-dependent manners. After 24-h stimulation, 10 nM PAF increased the levels of VEGF protein in astrocyte-conditioned medium by 1.3-fold. When the cells were subjected to hypoxia, the PAF-induced production of VEGF was enhanced by 6.7-fold as compared to the unstimulated cells incubated under normoxia. Dexamethasone was found to inhibit the enhanced VEGF production in response to the stimulation with PAF under hypoxia. We conclude that PAF induces VEGF gene expression in human astrocytes, and the PAF-induced increase in the expression of VEGF may modulate nervous tissue injury due to hypoxia.

15-Deoxy-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells.

Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of nuclear hormone receptor superfamily, and is known to play a role in various biological processes including inflammatory responses and adipocyte differentiation. CX3CL1/fractalkine is a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes. Endothelial cells produce fractalkine when stimulated with cytokines such as interleukin‐1 (IL‐1), tumour necrosis factor‐α and interferon‐γ (IFN‐γ). We herein report that 15‐deoxy‐n12,14 ‐prostaglandinJ2 (15d‐PGJ2), a PPAR‐γ agonist, inhibits the expression of fractalkine induced by IFN‐γ or IL‐1β in human endothelial cells. Agonist for PPAR‐α (WY14643) or PPAR‐γ (ciglitazone) did not inhibit the cytokine‐inducedfractalkine expression, and the effect of 15d‐PGJ2 maybe independent of PPAR. 15‐Deoxy‐D12,14 prostaglandin J2 also inhibited the adhesion of blood mononuclear cells to endothelial monolayers treated with IFN‐γ or IL‐1β. The data suggest that 15d‐PGJ2 regulates inflammatory reactions, at least in part, through the inhibition of fractalkine expression and leucocyte traffic through the endothelium.

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits IFN-γ-Induced Galectin-9 Expression in Cultured Human Umbilical Vein Endothelial Cells

Background: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-γ (IFN-γ). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-γ (PPAR-γ), and known to modulate the expression of various genes. Methods: We have studied the effect of 15d-PGJ2 on the IFN-γ-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture. Results: 15d-PGJ2 inhibited the IFN-γ-induced galectin-9 expression in a PPAR-γ-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-γ. 15d-PGJ2 partially inhibited IFN-γ-induced phosphorylation of STAT-1 in HUVEC. Conclusions: 15d-PGJ2 may regulate inflammatory reactions through the inhibition of galectin-9 expression.